α-Actinin 4 Links Vasopressin Short-Term and Long-Term Regulation of Aquaporin-2 in Kidney Collecting Duct Cells

Abstract

Water permeability of the kidney collecting ducts is regulated by the peptide hormone vasopressin. Between minutes and hours (short-term), vasopressin induces trafficking of the water channel protein aquaporin-2 to the apical plasma membrane of the collecting duct principal cells to increase water permeability. Between hours and days (long-term), vasopressin induces aquaporin-2 gene expression. Here, we investigated the mechanisms that bridge the short-term and long-term vasopressin-mediated aquaporin-2 regulation by α-actinin 4, an F-actin crosslinking protein and a transcription co-activator of the glucocorticoid receptor. Vasopressin induced F-actin depolymerization and α-actinin 4 nuclear translocation in the mpkCCD collecting duct cell model. Co-immunoprecipitation followed by immunoblotting showed increased interaction between α-actinin 4 and glucocorticoid receptor in response to vasopressin. ChIP-PCR showed results consistent with α-actinin 4 and glucocorticoid receptor binding to the aquaporin-2 promoter. α-actinin 4 knockdown reduced vasopressin-induced increases in aquaporin-2 mRNA and protein expression. α-actinin 4 knockdown did not affect vasopressin-induced glucocorticoid receptor nuclear translocation, suggesting independent mechanisms of vasopressin-induced nuclear translocation of α-actinin 4 and glucocorticoid receptor. Glucocorticoid receptor knockdown profoundly reduced vasopressin-induced increases in aquaporin-2 mRNA and protein expression. In the absence of glucocorticoid analog dexamethasone, vasopressin-induced increases in glucocorticoid receptor nuclear translocation and aquaporin-2 mRNA were greatly reduced. α-actinin 4 knockdown further reduced vasopressin-induced increase in aquaporin-2 mRNA in the absence of dexamethasone. We conclude that glucocorticoid receptor plays a major role in vasopressin-induced aquaporin-2 gene expression that can be enhanced by α-actinin 4. In the absence of vasopressin, α-actinin 4 crosslinks F-actin underneath the apical plasma membrane, impeding aquaporin-2 membrane insertion. Vasopressin-induced F-actin depolymerization in one hand facilitates aquaporin-2 apical membrane insertion and in the other hand frees α-actinin 4 to enter the nucleus where it binds glucocorticoid receptor to enhance aquaporin-2 gene expression.

Glucocorticoid Receptor Maintains Vasopressin Responses in Kidney Collecting Duct Cells

“…The infected cells were selected for stable knockdown with puromycin (Watson Biotechnology, Taiwan, 2.5 μg/ml) for two passages. GR mRNA was reduced to 43% (shGR1) and 45% (shGR2) of that in the non-target control, similar to previous values ( Ho et al, 2021 ).…”

“…In the control cells without glucocorticoid receptor knockdown, dDAVP induced a 22.5-fold increase in AQP2 mRNA compared to unity under the vehicle conditions ( Figure 1A , shCtrl, vehicle vs. dDAVP). The dDAVP-induced increases in AQP2 mRNA were diminished in the glucocorticoid receptor knockdown cells with two different small-hairpin sequences ( Figure 1A , shGR1 and shGR2, vehicle vs. dDAVP), confirming the effects of glucocorticoid receptor knockdown on the vasopressin-induced Aqp2 gene expression ( Ho et al, 2021 ). RNA-seq was performed to analyze the transcriptomes of these cells (shCtrl, shGR1, and shGR2) under the vehicle conditions (i.e., without dDAVP stimulation).…”

“…To identify pathways regulated by the glucocorticoid receptor for the vasopressin-induced Aqp2 gene expression ( Kuo et al, 2018 ), we compared transcriptomes of the mpkCCD cells with or without stable glucocorticoid receptor knockdown ( Ho et al, 2021 ). Figure 1A characterizes the responses of these cells to the vasopressin V2 receptor-specific analog dDAVP vs. vehicle.…”

“…In line with this, glucocorticoid receptor agonist dexamethasone increases the vasopressin-induced Aqp2 gene expression in the collecting duct mpkCCD cells ( Kuo et al, 2018 ). The stable glucocorticoid receptor knockdown blunted the vasopressin-induced Aqp2 gene expression in the cells ( Ho et al, 2021 ). In the current study, we found that the transcriptome was significantly altered in the stable glucocorticoid receptor knockdown cells that do not respond to vasopressin ( Figure 1 ).…”

“…We recently found that dexamethasone enhanced the vasopressin-induced Aqp2 gene expression in a time- and dose-dependent manner in the mpkCCD cells, a collecting duct principal cell model that expresses all necessary molecular components for vasopressin signaling, Aqp2 gene expression, and trafficking ( Yu et al, 2009 ; Rinschen et al, 2010 ; Xie et al, 2010 ; Khositseth et al, 2011 ; Schenk et al, 2012 ; Loo et al, 2013 ; Kuo et al, 2018 ). Glucocorticoid receptor knockdown blunted the vasopressin-induced Aqp2 gene expression in the cells ( Ho et al, 2021 ). The effects of the glucocorticoid receptor involve α-actinin 4, an actin-bundling protein and a transcription co-activator of the glucocorticoid receptor ( Honda, 2015 ; Zhao et al, 2017 ).…”